mise the usefulness of the protein ladder presented here.
It is not clear how the cycle of reduction/oxidation described here resulted into the formation of monomer, dimer, trimers, and other oligomers in somewhat similar proportions. MB-1TrpRH has only 2 Cys; therefore, only one intramolecular bridge may form in a monomer. The protein must form bridges with other monomers (i.e., the bridges cannot be intramolecular) to form dimers or higher oligomers. Since those bridges involve Cys side chains that are expected to be in core positions in the monomers, it appears that formation of the ladder involves a drastic change in conformation for MB-1TrpRH subunits in dimer and higher oligomers. Interestingly, the proteins remain soluble, even after extensive cross-linking, which indicates that somehow the core side chains are protected in the new cross-linked conformation. Further measurements are in progress to elucidate this phenomenon.
We have shown here the synthesis of various oligomers of a mutant proteins produced by a single bacterial clone. After a cycle of reduction/oxidation of the disulfide bridges, the oligomers range in sizes from 11 to approximately 125 kDa, and migrate as a ladder on an SDS-PAGE gel. Such a ladder is convenient in that it is made of the same proteins with sizes that are multiples of 11 kDa.